Controls



Feb. 21, 1956 H. c. MURRAY ET AL 2,735,800

STEROIDS Filed July 5, 1952 20 Sheets-Sheet l IO N 0 1 ID o o (\l 0 Z0'7 2 9 5 m o z 69 G) O g z m a [L U] I I Q 8 g 2 LL :0

.I ll 1 m o o 9 NOISSIWSNVHL lNBOHHd HERBERT c. MURRAY DUREY H. PETERSONINVENTORS ATTOR NE Y Feb. 21, 1956 Filed July 5 1952 2O Sheets-Sheet 2 Ni w z 0 n: m -0 (no on: o. m x 9... M:

HERBERT c. MURRAY DUREY PETERSON INVENTORS ATTOR NE Y STEROIDS 20Sheets-Sheet 3 m 6? T20 Z ozmnommm 00 00mm 000m 00mm 000m 00m- 009 00hDUREY H PETERSON INVENTORS BY W %M ATTORNEY I A R R U M C T R E B R E HNOISSIWSNVHJ.

Feb. 21, 1956 H. c. MURRAY ET AL Filed July 5, 1952 Feb. 21. 1956 H. C.MURRAY ET AL STEROIDS Filed July 5, 1952 ll KETOPROGESTERONE- IDENTITYoH CONTROL CO 108.3 +DHP-l08.3

80g of 0am Jam fie wed 3|9-DHP-I08.3 326-MIU-l26 DHP-IO8.3=Cr0 OXIDA-nou 0F CMPD U=11I(8O7) 0ONTROL= AUTHENTIC SAMPLE ll-KETOPROGES- TERONE(80y) FIG. 4

2O Sheets-Sheet 4 CMPD S BIOCONVERSION BY RH|76 PROPYLENEGLYCOL-TOLUENEs48=4s HR. FERMENTATION 0F com ouuo 8(3007) S48+OONTROLSz CONTROL$=COMPOUND F(l6 COMPOUND E067) COMPOUND 562 HERBERT C.MURRAY N DUREY H.PETERSON I E ms BY W 51 ATTORNEY Feb. 21. 1956 H. c. MURRAY ET AL2,735,800

STEROIDS Filed July 5, 1952 20 Sheets-Sheet 5 CMPD S BIOCONVERSION BYIT-(PHYDROXYPROGESTER- RHI76 ONE CONVERSION SIGHRS. CONTROLS S48HRSHOP-l6 CONTROLS+ S28 HRS. S40 HRS. HOP-241 P HOP-2Q v H ET SEQ.

PROPYLENEGLYCOUTOLUENE HOP|6=l7-(!-HYDROXYPRO- GESTERONE (300yM6 HRSFERMENTATION HOP*24=DITTO,24 HRS. FER- MENTATION CONTROLS:

n-a-nvorzoxvmosssrm ONE(CA.24O mmv- DROXYPROGE TERONE, DHP-95.4(60y)PROPYLENEGLYCOL- TOLUENE S=GOMPOUND s FERMENTATION 320,); SUBSCRIPTHOURS OF FERMENTATION CONTROLS:

COMPOUND F (I67) COMPOUND E (167) COMPOUND s (327) coRTacosTERoNE (327)Fla 6 FIG] HERBERT C. MURRAY DUREY H. PETERSON INVENTORS BY 2 g, I MATTORNEY Feb. 21, 1956 2,735,800

H. C. MURRAY ETAL STEROIDS Filed July 5. 1952 20 Sheets-Sheet 6 M 0CHROMATOGRAM 22 coNTRoLs [FRAc'noNs RACTIONSIF A1203 CHROMATOGRAM 223l9-DHP-lll l 349-$HE37,38 349-SHE- 7,38

ALL FRACTIONS APPLIED AT ALL FRACTIONS APPLIED AT I607 LEvEL I607 LEVELMIXED CONTROL:

COMPOUND E (I67) DIHYDROXYPROGESTERONE (DHP 95.4) (301 (DHP 95.4)(30y)F= COMPOUND F CONTROL F= COMPOUND FCONTROL THE 'Al O3 CHROMATOGRAM THEM203 CHROMATOGRAM OF ZBB-HCM'ZMUTQ HO, OF ZGG-HCM-ZMUYGHO, PROGESTERONE)PROGESTEROND) INVENTORS HERBERT c. MURRAY BY DUREY H. PETERSON MIXEDGONTROLI COMPOUND E (l6 7') DIHYDROXYPROGESTERONE FIG. 8

Feb. 21, 1956 H. c. MURRAY ETAL 2,735,800

STEROIDS Filed July 5, 1952 20 Sheets-Sheet 7 PROGESTERONE suocou-PROGESTERONE BIOCON- VERSION VERSION CONTROLS OCI ocz PROG.

RI P? K P3 P4 349-S.HE-40(P4) PROPYLENEGLYCOUTOLUENE P4=PROGESTERONEFERMEN- TATION (300 CONTROLS:

DIHYDROXYPROGESTERONE 266-HCM'l99'OCi AND 062 363'MlU-2lCARBlTOL-METHYLCYCLO- HEXANE PROGESTERONE FER- 0C2 MENTATIONS (5007)PROG.=PROGESTERONE cou- TROL (807) (DHP 95.4) 68 II'Q-HO, PRO ESTERONEFIG. I!

HERBERT C. MURRAY T DUREY H. PETERSON INVEN 0R8 ATTOR NE Y Feb. 21, 1956H. c. MURRAY ETAL 2,735,800

STEROIDS Filed July 5, 1952 20 Sheets-Sheet 8 PROGESTERONE BIOCON-VERSION CONTROL CONTROL 0 Pl P2 8 266HCM-2l2/3OOB AND l99/3OOT349-SHE-44Pl AND 44PZ PROPYLENEGLYCOL-TOLUENE PI=PROGESTERONE FERMEN-T1830? (HCM-2l2/3OOB) P2=PROGESTERONE FERMEN- TATION (HCM-l99/3OOT) (300CONTROLS:

a=| l-O-HO, PROGESTERONE 7 7-. .4 B =DIHYDROXYPROGESTER- ONE (DHP 95.4)(307) F l6. l2

DOC PROGESTERONE CON" VERSION 204DR CORT FROG DOC 205PP 266'HCM204DR AND205PP CARBITOL'METHYLCYCLO- HEXANE 204DR=|I*DESOXYCORT|CO$- TERONEFERMENTATION (3207) DOCHIDESOXYCORTICOS" TERQNE CONTRQL (64y) CORT=CORT'COSTERONE CONTROL (64y) PROG.= PROGESTERQNE CONTROL (807) 205PP=PROGESTERONE FERMENTATION (320)!) Fla/3 HERBERT C. MURRAY NVENTORs DUREYH. PETERSON 1 M ATTORNEY Feb. 21, 1956 Filed July 5, 1952' [Fa-HO,PROGESTERONE BiOCONVERSlON CONTROL$+ ass-mamas HPZ 349-sH-22 PROPYLENEGLYGOL- TOLUENE HP2=n-a-Ho moses TERONE FERMENTA- now (320 CONTROLS.

DIHYDROXYPROGESTER- our: (on? 914M60 GORTIGOSTERONE (647) FIG. 14

H. c. MURRAY ET AL 2,735,800

STEROIDS 20 Sheets-Sheet 9 CMPD S-ACETATE BIOCON- VERSION CONTROLS+209-SA ZOQ'SA l 266"HCM-209SA &349-SH PROPYLENE GLYCOL-TOLUENE 209'SA=COMPOUND S'ACE' TATE FERMENTATION (3207) CONTROLS: COMPOUND F06 E0 7)3(641) FIG. 15

HERBERT c. MURRAY V DUREY H. PETERSON IN ENTORS ATTOR NE Y Feb, 21, 1956H, c. MURRAY ET AL 2,735,800

STEROIDS Filed July 5. 1952 20 Sheets-Sheet 10 D06, DOCA BIOCONVERSIONCOMPOUND s BIOCONVERSION CONTROLS+ BY RH CONTROLS+-1 1 5A2 52 A2 s2HCM-l98D2+ DAZ 349 -$HE2I PROPYLENEGLYCOL-TOLUENE D2 II-DESOXYCORTICOSTER- ONE (320 266'HCM-I98 52 AND 5A2 DA2 II'DESOXYCORUCOSTER- HE' 25 ONE ACETATE (3207) PROPYLENEGLYCOL-TOLUENECONTROLS D2 S2=COMPOUND S FERMENTA- CONTROLS 0A2 TION (3207) CONTROLS iCOMPOUND F (I67) 5 2 CQMPQUND s-ACETATE EH6 FERMENTATEON (320 $054CONTROLS! COMPOUND F (16,) coancosrsnous (647) 5?:

F16, [6 FIG. I?

HERBERT c. MURRAY To DUREY H. PETERSON Rs A TORNEY Feb. 21, 1956 H. c.MURRAY ETAL 2,735,800

STEROIDS Filed July 5. 1952 20 Sheets-Sheet 11 S 8 S'ACETATE BIOCON'CMPD S BIOCONVERSION M VERSION CONTROLS BY 6 l CONTROLS 2ss-HcM-ma-s AND5A4 349-sHE-26- ZSS-HCM-ZITMUS a PROPYLENEGLYCOLTOLLIENE 9$HE-4a S=COMPOUND S E PROPYLENEGLYCOL TOLUENE TATION 1 noowouwo s FERMEN-ESME32%&?JJ

r CONTROLSzCOMPOUND F C0NTROLS:COMPOUND F061) COMPOUND 5%:3?

COMPOUND H COMPOUND 5(32 compouuo 8(6 Fla/8 F/6./.9

HERBERT C. MURRAY INVENTORS DUREY H. PETERSON ATTORN E Y Feb. 21, 1956H. c. MURRAY EIAL 2,735,800

STEROIDS Filed July 5. 1952 20 Sheets-Sheet l2 ANDROSTENEDIONE BIOGON-ANDROSTENEDIONE BIOGON- VERSION VERSION MAND AR 000 PROG 5 AND AR DOGPROG S ARa349sHE- 266-HGM' 1 ma -SH 70.3 70.3 PROPYLENEGLYGOL-TOLUENECARBITOL-METHYLCYCLO- AND=ANDROSTENEDIONE cou- HEXANE TROL 1)AND=ANDROSTENEDIONE AR= ANDROSTENEDIONE FER- CONTROL (807) MENTATION(500 AR=ANDROSTENEDIONE FER- ooc u- DESOXYGORTIGOS- MENTAT|0N 7 TERONEGONTROL(64 DOG=Il-DESOXYGORTICOS- PRQG=PROGESTERONE CON- TERONECONTROI-(SW) TROL (80y) PROG=PROGESTERONE con- 5 =com=ou-0 s CONTROLTROL (807) (327) S=COMPOUND s CONTROL HERBERT C. MURRAY NTOR DUREY H.PETERSON M ATTORNE Y 2 1956 H. c. MURRAY ET AL 2,735,300

' HERGOSEERONE BlOCON- VERS|0N\BY RH|76 Filed July 5. 1952 CHOLESTENONEBIOCON- VERSION BY RHI76 GHOL DOG ERG 000 OR PROG OR PROG 266 HOM-ZISORAND, 349- SHE-70.2

CARBiTOL-METHYLCYCLO- HEXANE CHOL= CHOLESTENONE CON- TROL (8O CR=CHOLESTENONE FER- MENTATION (500 DOC=1I- DESOX YCORTICOS- TERONE CONTROL(64 PROG=PROGESTERONE cou- TROL (64y) 266*HCM-2l9'OR AND 349' SHE-70.1CARBlTOL-METHYLCYGLO'" HEXANE OR=ERGOSTERONE FERMEN- TATION (5007)ERG=ERGOSTERONE CON" TROL (807) DOC=II-DESOXYCORTICOS' TERONE CONTROL(64 PROG= PROGESTERON E CON ROL (647) HERBERT C. MURRAY INVENT DUREY H.PETERSON 0R5 ATTOR NE Y Feb. 21, 1956 H. c. MURRAY EI'AL 2,735,800

STEROIDS Filed July 5. 1952 20 Sheets-Sheet l4 ERGOSTEROL BiOGONVER-PROGESTERONE CONVERSION,

SiON VEGETATIVE INOCULATION,

CONTROLS SIMPLE MEDIUM CONTROLS ER EB CONTROLS VEG +VES5. CONTROLS 53.2PROPYLENEGLYCOL-TOLUENE VEG=PROGESTERONE FER- MENTATION CONTROLS!PRORYLENEGLYCOL -ToLueNs 96% PROGESTERWE 266- era-219 ER =ERGOSTEROLFERMEN- TAT'ON 5007) DIHYDROXYPROGESTERONE CONTROLS; (301)H-DESOXYOORTICOSTERONE v ERGOSTEROL 9s COMPOUND s (3%) 25 DUREY H.PETERSON Feb. 21, 1956 H. C. MURRAY ET AL STEROIDS Filed July 5. 1952PROGESTERONE BIOCON- VERSION BY MUCORALES CONTROLS Ml M4 M5 3l4AW-64-7la 349-SHE-47 PROPYLENEGLYCOL-TOLUENE MI=MIIP6 PROGESTERONE FERMENTATION320 M4=M4IP6 PROGESTERONE FERMENTATION (320, M5=M5IP6 PROGESTERONEFERMENTATION (320 CONTROLS:

HG-HO,PROGESTERONE (so DIHYDROXYPROGES- TE ONE(DHP 95.4)(30r) 20Sheets-Sheet 5 PROGESTERONE B'OGON- VERSION BY MUCORALES CONTROLS M2 M3M sg A B A. .O

PROPYLENEGLYCOL" TO LUENE M2= MZ/PG FERMENTATION mag/P6 FERMENTATION M6=Me/Ps FERMENTATION (240v) CONTROLS:

na-Ho PROGESTERONE A ag OIHYDROXYPROGES' ONE (DHP 95.4)(307) B-lIQ'HO,PROGESTERONE (6?) nae? HERBERT c. MURRAY INVENmRS DUREY H.PETERSON ATTORNEY 1 Feb. 21, 1956 I Filed July 5, 1952 PROGESTERONEBiOCONVER- SION BY MUCORALES CONTROLS PROPYLENEGLYCOLTQLUENE M7 M7/P6FERMENTATION M8= M8/P6 FERMENTATION CONTROLS:

H-a" H0, PROGESTERONE DIHY DROXY FROG ESTE RONE H. C. MURRAY ETAL 20Sheets-Sheet l6 PROGESTERONE BIOCONVER- SION BY MUOORALES RSGONTROLSPROPYLENEGLYCOL TOLUENE R I R l/P6 FERMENTATION R%; R2/P6 FERMENTATION20 R6 RZ/PS FERMENTATIQN (3207) CONTROLS: "(1'' H0, PROGESTERONEDIHYDROXYPROGESTERONE (DHP 95.4)(50 HERBERT c. MURRAY INVENTORS DUREY H.PETERSON ZMM Feb. 21, 1956 Filed July 5, 1952 PROGESTERONE' BIOCONVER-SION BY MUGORALES CONTROL PROPYLENEGLYCOL-TOLUENE R3= R3/P6 FERMENTATIONR4=R4 P6 FERMENTATION CONTROLS;

O =H'Q'HO, PROGESTER' one (301) DIHYDROXY- PROGESTERONE (DHP 95.4) (307)B=|l"a-Ho, PROGESTER' ONE(607) FIG. 30

H. C. MURRAY ET AL STEROIDS 20 Sheets-Sheet l7 PROGESTERONE BIOOONVER-SION BY MUCORALES R5 R7 R8 CONTROLS 3|4-AW-64-7l 8 349-SHE-50PROPYLENEGLYCOL-TOLUENE Rigggl6 FERMENTATION R7= R7/P6 FERMENTATION(3207) R8=R8IP6 FERMENTATlON NTR 0 PRO- %EST% R Oh l:E( 64 (soDIHYDROXYPRO sTE ONE DHP 95.4) INVENTORS FIG. 3/ HERBERT G. MURRAY BYDUREY H. PZTERSON ATTOR NE Y F 1. 1956 H. c. MURRAY ET AL 2,735,300

STEROIDS Filed July 5, 1952 20 She s-S ee 18 00c BIOOONVERSION BY w g iMUCORALES MUCORALES mzm'rcc IZO'IA) goumms M CONTROLS M l M? +M2CONTROLS PROPYLENEGLYCOL TOLUENE Ml M V0006 FERMENTATION M2= MZ/DOCGFERMENTATION M3=M3IDOC6 FERMENTATION CONTROLS: MIXTURE OF COMPOUND F(I67) COMPOUND E (I6 CORTICOSTERON (64y) PROPYLENEGLYCOL-TOLUENEM2=M2IDOC6 FERMENTATIO CONTROLS: MIXTURE 0F COMPOUND F (I67) com ouuo E(I67) 90 COSTERONE (64y) HERBERT c. MURRAY N DUREY H. PETERSON I VENTORSBY 7 W A TTORNEY Feb. 21. 1956 Filed July 5, 1952 MucoRALEs mums-1851)H. C. MURRAY ET AL STEROIDS CONTROLS: MIXTURE OF COMPOUND F (I67)COMPOUND E (l6 CORTICOSTERO 20 Sheets-Sheet l9 PROGESTERONE CONVER- SIONBY BEER FILTRATE CONTROL 82 R CONTROL= ROGESTERONE SI=FERMENTATION BYDI- LUTED BEER FILTRATE PH 6.76 v

S2= LIKE SI BUT PH 4.65 R FERMENTATEON BY MY- OELIUM BF=FERMENTATON BYUN- DILUTED BEER FILTRATE PH 4.! GARBITOL- METHYLCYOLO- HEXANE HERBERT aMURRAY ENTORS DUREY H. PETERSON BY 5149 7 TORNEY Feb. 21, 1956 H. c.MURRAY ET AL 2,735,800

STEROIDS Filed July 5, 1952 20 Sheets-Sheet 20 ll-(I- HO, PROGESTERONECON- VERSION BY RH I76 CONTROLS 72 +72 CONTROLS HCM- 9 & 349'SHE'72PRQPYLENEGLYCQL-TQLUENE 72 Ila-H0, PROGESTE RONE FE RMENTATION (320CONTROLS:

Ila-HO, PROGESTEROPE (60 7) omvoaoxvpaoess'renous (DHP 95.4)(307) FIG.36

HERBERT O. MURRAY INVENToRs DUREY H. PETERSON ATTORN E Y

1. IN AN AEROBIC MYCELIAL FERMENTATION PROCESS OF INTRODUCING OXYGENINTO A STEROID, THE STEPS COMPRISING OXYGENATING A STEROID WITH AT LEAST0.5 GRAMS, ON A DRY BASIC, OF MYCELIUM PER GRAM OF STEROID UNTIL THEWEIGHT RATIO OF THUS OXYGENATED STEROID TO RESIDUAL STARTING STEROID ISAT LEAST FOUR TO ONE AND THEN SEPARATING AND ISOLATING THE RESULTINGOXYGENATED STEROID.